A20 ubiquitin ligase-mediated polyubiquitination of RIP1 inhibits caspase-8 cleavage and TRAIL-induced apoptosis in glioblastoma

Abstract

The TNF-related apoptosis-inducing ligand (TRAIL) apoptotic pathway has emerged as a therapeutic target for the treatment of cancer. However, clinical trials have proven that the vast majority of human cancers are resistant to TRAIL apoptotic pathway-targeted therapies. We show that A20-mediated ubiquitination inhibits caspase-8 cleavage and TRAIL-induced apoptosis in glioblastoma through 2 signaling complexes. A20 is highly expressed in glioblastomas and, together with the death receptor 5 and receptor-interacting protein 1, forms a plasma membrane-bound preligand assembly complex under physiologic conditions. Treatment with TRAIL leads to the recruitment of caspase-8 to the plasma membrane-bound preligand assembly complex for the assembly of a death-inducing signaling complex. In the death-inducing signaling complex, the C-terminal zinc finger (Znf) domain of the A20 ubiquitin ligase mediates receptor-interacting protein 1 polyubiquitination through lysine-63-linked polyubiquitin chains, which bind to the caspase-8 protease domain and inhibit caspase-8 dimerization, cleavage, and the initiation of TRAIL-induced apoptosis in glioblastoma-derived cell lines and tumor-initiating cells.

Significance: These results identify A20 E3 ligase as a therapeutic target whose inhibition can overcome TNF-related apoptosis-inducing ligand resistance in glioblastoma and thus have an impact on ongoing clinical trials of TNF-related apoptosis-inducing ligand-targeted combination cancer therapies.

Figure 1

TRAIL-induced formation from the PLAC to the DISC. A, Normal brain and glioblastoma tissues were examined by IB using antibodies as indicated (left). The molecular weights were indicated to the right of the panels. Actin was used as a loading control. B, DR5-associated PLAC and DISC were isolated from LN443 and LN71 cells treated with mixed Flag-TRAIL and Flag antibody for 15 min and analyzed by IB with cell lysates as controls. C, Size exclusion fractions from LN443 and LN71 cells untreated or treated with 100ng/ml TRAIL for 15 min were analyzed by IB. The elution position of molecular weight markers in kDa are indicated at the top of the panels. D, The PLAC was isolated from the pooled high molecular weight fraction 42–50 and low molecular weight fraction 62–70 of LN443 cells and examined by IB. The input was included, showing the protein loading. E, Subcellular cytosol (Cytos), membrane (memb), and nuclear (Nucl) fractions from LN443 were examined by IB with antibodies to glyceraldehyde-3-phosphate dehydrogenase (GAPDH), c-Jun and epidermal growth factor receptor (EGFR) as loading controls, respectively, for cytosol, nuclear and membrane fraction. F, Subcellular cytosol and membrane fractions from LN443 and LN71 were subjected to IP using a DR5 antibody with IgG included as a negative control and examined by IB for the presence of the proteins as indicated (left).

Figure 2

A20 inhibits caspase-8 cleavage and TRAIL-induced apoptosis. A, TRAIL-resistant LN443 cells were transfected with A20, TRAF2 and scrambled siRNA, treated with TRAIL for 24 hr, and examined for cell death (points: means; bars: SE; n=6; ***, p < 0.001). B, The transfected cells were treated with 100ng/ml TRAIL and examined by caspase-8 enzymatic activity assay (points: means; bars: SE; n=6; ***, p < 0.001; NS, no significance). C, Apoptotic cell death was observed under phase contrast microscopy in A20 siRNA but not scrambled and TRAF2 siRNA transfected cells. D, LN443 cells were transfected with siRNA, treated with 100 ng/ml TRAIL and examined by IB for the knockdown of the proteins and the cleavage of caspase-8 and RIP1. Non-transfected TRAIL-sensitive U343MG cells were included as the control for caspase-8 and RIP1 cleavage. E, The PLAC and the DISC were isolated from the LN443 cells transfected with siRNA and examined by IB for caspase-8 cleavage. F, LN443 cells transfected with A20 and RIP1 siRNA were treated or not with 100 ng/ml TRAIL in the absence or presence of the caspae-8 inhibitor z-IEDT and examined by IB for caspase-8 cleavage.

Figure 3

A20 E3 ligase inhibits caspase-8 cleavage. A, LN71 stable clones expressing A20 wt, OTU and Znf mt and empty vector were treated with 100 ng/ml TRAIL for 24 hr and examined for cell death (points: means; bars: SE; n=6; ***, p < 0.001). B, LN71 stable clones were treated with 100 ng/ml TRAIL in the absence or presence of z-IEDT for caspase-8 activity (points: means; bars: SE; n=6; ***, p < 0.001). C, The PLAC and DISC were isolated from LN71 stable clones through IP using Flag-TRAIL and Flag antibody and examined by IB for the presence of the proteins as indicated (left). D, LN71 stable clones were treated with TRAIL for the times indicated (top) and examined by IB for A20 expression and cleavage of RIP1.

Figure 4

A20 E3 ligase mediates RIP1 K63-linked polyubiquitination. A, In vitro ubiquitination was carried out in a reaction consisting of the components as indicated (top) with ubiquitinated RIP1 detected by an avidin antibody on IB. B, In vivo ubiquitination was performed by transfecting HEK293T cells with the vectors encoding Flag-RIP1, A20 and HA-UB mts and detecting ubiquitinated RIP1 by IB using myc and HA antibody. C, In vivo ubiquitination in U87MG cells were conducted by transfecting the cells with the vectors encoding HA-UB and mts, treating the transfectants with 100 ng/ml TRAIL for 1.5 hr, isolating RIP1 under denaturing condition and detecting ubiquitinated RIP by IB using antibodies to HA and RIP1. The number represents the quantification of the density. D, The PLAC and DISC were isolated from LN443 cells through IP using Flag-TRAIL and Flag antibody. Ubiquitinated RIP1 was detected by overexposure of IB using a RIP1 antibody. E, RIP1 was purified through IP under denaturing conditions from the PLAC and DISC as in (D) and ubiquitinated RIP1 was detected by IB using an UB antibody. F, RIP1 isolated from LN443 cells as in (E) was examined by IB using antibodies specific to K63-linked polyUB chain and RIP1. G, The PLAC and DISC were isolated from LN71 clones expressing A20 wt, OTU and Znf mt through IP as in (F) and examined by IB using antibodies specific to K63-linked polyUB chain and RIP1.

Figure 5

K63-linked UB chain binds to caspase-8 and inhibits its cleavage. A, Caspase-8 was purified using its antibody through IP from LN443 cells after treatment with 100 ng/ml TRAIL and examined by IB for UB-conjugated RIP1. B, In vivo Fv-caspase-8 binding assay was carried out in LN443 cells. The cell line was either treated or not with 100 ng/ml TRAIL for the times indicated (top) and then subjected to subcellular fractionation. RIP1 was isolated through IP and caspase-8 binding to the RIP1 K63-linked polyUB chain was detected by IB. C, In vitro caspase-8 binding to UB was examined by Fv-caspase-8 (left) and caspase-8 protease p18 subunit pull down (right), in which Fv-caspase-8 and p18 subunit bound beads were incubated with the mono-UB, K63, K48-linked and linear polyUB protein. Unbound (U) were washed off the beads and bound (B) proteins were eluted and identified by IB. * indicates a non-specific (NS) band. D, Nickel column pull down assay was carried out by incubating a His-K63 polyUB chain labeled nickel column and Fv-caspase-8 in various concentrations as indicated (top). The column was washed and the caspase-8 and His-K63 polyUB chain were eluted and examined by IB. E, In vitro caspase-8 cleavage assay was performed by incubating mono-UB and polyUB chains with Fv-caspase-8 for 2 hr, then adding Fv ligand AP20187 and detecting caspase-8 cleavage in the reactions by IB.

Figure 6

The PLAC and DISC are identified in glioblastomas. A–D, Glioblastoma tumor tissues and derived CD133+ cells (EH 091112) were subjected to size exclusion assay (A, B) and subcellular fractionation (C, D) and then examined by IB for the presence of the proteins as indicated (left). E, The PLAC was isolated using Flag-TRAIL from the pooled high molecular weight fraction 42–50 and low molecular weight fraction 62–70 of CD133+ cells (EH 091112) and examined by IB. The input was included to show the protein loading. F–G, The PLAC and DISC were isolated from CD133+ cells (EH 091112, 091106) through IP using Flag-TRAIL and Flag antibody and examined by IB for the presence of the proteins (F) and the RIP1 ubiquitination (G).

Figure 7

A20 protects the tumor-initiating cells from TRAIL-induced apoptosis. A–C, CD133+ cells (EH 091112, 100113, 091217 and 091106) were transfected or not with A20 and scrambled siRNA, treated or not with 100 ng/ml TRAIL, and examined for cell death (A), IB (B) and caspase-8 activity (C). D, EH 091112 cells were transfected with the lentiviral vectors encoding the A20 wt, OTU and Znf4 mt, treated or untreated with 100 ng/ml TRAIL and examined for cell death and caspase-8 activity Each of the experiments was repeated eight times (points: means; bars: SE; n=6; ***, p < 0.001). E, A two-complex model of A20 E3 ligase-mediated inhibition of caspase-8 through RIP1 polyubiquitination.